JVI Accepted Manuscript Posted Online 17 February 2016 J. Virol. doi:10.1128/JVI.02878-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Linear epitopes in A27 are targets of protective antibodies induced
2
by vaccination against smallpox
3 4
Running title: Protective linear epitopes of vaccinia virus A27
5 6
Thomas Kaever1, Michael H. Matho2, Xiangzhi Meng3, Lindsay Crickard1, Andrew Schlossman2,
7
Yan Xiang3, Shane Crotty1,4, Bjoern Peters1, Dirk M. Zajonc2,5^
8 9 10 11 12 13 14 15 16
1
Division of Vaccine Discovery, 2Division of Cell Biology, La Jolla Institute for Allergy and
Immunology (LJI), La Jolla, CA 92037, USA. 3
Department of Microbiology and Immunology, University of Texas Health Science Center at
San Antonio, San Antonio, TX 78229. 4
Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA
92037, USA 5
Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University,
9000 Ghent, Belgium
17 18
^ Corresponding author:
19
Dirk Zajonc
20 21
Abstract: 225 words
22
Full text: 7,496 words
1
23
Abstract
24
Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax
25
smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and
26
binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their
27
protective capacity and their interaction with A27, we generated and subsequently characterized
28
7 murine monoclonal antibodies (mAbs), which fell into 4 distinct epitope groups. Three groups
29
(I, III and IV) bound to linear peptides, while group II only bound to VACV lysate and
30
recombinant A27, suggesting it recognized a conformational and discontinuous epitope. Only
31
group I antibodies neutralized MV in a complement-dependent manner and protected against
32
VACV challenge, while a group II mAb protected partially but did not neutralize. The epitope
33
for group I mAbs was mapped to a region adjacent to the GAG binding site and suggest that
34
group I mAbs could potentially interfere with cellular adhesion of A27. We have further
35
determined the crystal structure of the neutralizing group I mAb 1G6, as well as the non-
36
neutralizing group IV mAb 8E3 bound to the corresponding linear epitope-containing peptides.
37
Both light and heavy chains of the antibodies are important in binding their antigens. For both
38
antibodies the L1 loop seems to dominate the overall polar interactions with the antigen, while
39
for mAb 8E3, the light chain generally appears to make more contacts with the antigen.
40 41
Importance
42
Vaccinia virus is a powerful model to study antibody responses upon vaccination, since
43
its use as the smallpox vaccine led to the eradication of one of the world’s greatest killers. The
44
immunodominant antigens that elicit the protective antibodies are known, yet for many of these 2
45
antigens little information about their precise interaction with antibodies is available. In an
46
attempt to better understand the interplay between the antibodies and their antigens, we have
47
studied a panel of anti A27 antibodies from generation, functional characterization to the
48
interaction with the epitope using X-ray crystallography. We identified one protective antibody
49
that binds adjacent to the heparan sulfate binding site of A27, likely affecting ligand binding.
50
The analysis of antibody-antigen interaction supports a model in which antibodies that can
51
interfere with the functional activity of the antigen are more likely to confer protection than those
52
that bind at the extremities of the antigen.
53 54 55
Introduction Inoculation with vaccinia virus (VACV) elicits neutralizing antibodies against major
56
antigens, including A27, A33, B5, D8, H3, and L1 on both the extracellular enveloped virus (EV)
57
and the intracellular mature virion (MV or IMV), conferring protection against smallpox (2, 6, 7,
58
16, 26). As a result, wide-spread vaccination against smallpox (variola virus, VARV) led to the
59
first eradication of a viral pathogen from nature (11). Among the major immunodominant
60
antigens of the IMV, A27, H3, and D8 are adhesion molecules that bind to the
61
glycosaminoglycans heparan sulfate (A27 and H3) and chondroitin sulfate (D8) (13, 14, 18, 20).
62
We have previously shown that anti-D8 antibodies can prevent binding of D8 to chondroitin
63
sulfate. Besides its binding to heparan sulfate, little is known about the function of H3 (18).
64
However, human antibodies that target H3 in combination with those that target B5 provide
65
significantly better protection than either antibody by itself and are promising for the treatment
66
of smallpox infections in human (22). Since the general human population lacks protection
3
67
against smallpox due to the cessation of smallpox vaccination, protective antibodies can be used
68
to treat infected patients. While neutralizing anti-A27 antibodies protect against infection, they
69
only represent a minor component of the Dryvax vaccine induced immune response (10).
70
A27 is a homo-trimeric extracellular protein that is attached to the viral membrane by
71
binding to the transmembrane protein A17 through its C-terminal leucine zipper domain
72
(residues 80-101). The GAG binding site is located at the N-terminus, downstream of the signal
73
sequence (residues 21-30) (12, 30). The central region of A27 consists of a coiled coil domain
74
(residues 43-84), which is used to interact with the membrane fusion suppressor protein A26
75
through intermolecular disulfide bond formation (Cys71, Cys72). The crystal structure of an N-
76
terminal fragment of A27, containing the heparan sulfate binding site and coiled coil domain
77
(residues 21-84) had recently been determined, however only the central fragment (residues 47-
78
84) is ordered, suggesting flexibility of the N-terminal GAG binding domain (5). The A27
79
structure illustrates the complexity and antiparallel nature of the A27 homo-trimer, yet structural
80
information about the N-terminal and C-terminal extremities are missing.
81
In this study we have produced a panel of anti-A27 antibodies through immunizing mice
82
with VACV. We have identified 4 antibody groups based on cross-blocking experiments and
83
identified the epitope using a peptide/protein ELISA. Group I, II, and IV antibodies recognized
84
both VACV lysate as well as synthetic peptides, suggesting that the epitope of these antibodies
85
can be recapitulated using linear peptides. We have further determined the crystal structure of a
86
protective antibody 1G6 (group I), and a non-protective antibody 8E3 (group IV) in complex
87
with their respective linear peptide epitopes, which are located at the N- and C-terminal
88
extremities of A27, shedding light onto the structural basis of A27 recognition.
4
89
Materials and Methods
90
Viruses and antibodies. VACVWR stocks were grown on HeLa cells in T175 flasks and
91
infecting at a multiplicity of infection of 0.5. Cells were harvested at 60 h and virus was isolated
92
by rapidly freeze-thawing the cell pellet three times in a volume of 2.3 ml RPMI plus 1% fetal
93
calf serum (FCS). Subsequently, cell debris was removed by centrifugation. Clarified supernatant
94
was frozen at -80°C as stock. VACVWR stocks were titered on Vero cells (~2 x 108 PFU/ml).
95
VACVACAM2000 was obtained from the CDC. Monoclonal antibodies used in the study are: anti-
96
H3 #41, anti-D8 Ab12.1, anti-L1 M12B9, anti-A27 1G6, 12G2, 8H10, 6F11, 4G5, 12C3 and 8E3.
97
Hybridoma generation and characterization.
98
The hybridomas were generated as described previously (32). In brief, a six-week old BALB/c
99
mouse was infected intranasally with 5 × 103 plaque-forming-unit (PFU) of VACV WR. Seven
100
weeks after the infection, the mouse was injected intravenously with 7 × 107 PFU of UV-
101
inactivated WR virus. Three days afterwards, the spleen of the mouse was harvested for
102
hybridoma generation. Hybridomas that secret anti-A27 antibodies were identified with
103
immunofluorescence assays of HeLa cells infected with wild-type or A27-deletion VACV strains
104
(31).
105
chromatography and purity was assessed by reducing and non-reducing SDS PAGE. All
106
experiments were performed using purified antibodies.
107
A27 expression and purification.
108
A27L with a C-terminal hexahistidine tag was cloned into pET15b (Invitrogen) and transformed
109
into CodonPlus BL21 cells (Agilent). After culture growth reached an OD600 of 0.6, A27
110
expression was induced by induction with 1mM IPTG at 37 °C for 4 h. Cells from typically 1 L
IgG’s were purified from hybridoma culture media using protein G affinity
5
111
were spun down (10 min x 4000 g) and resuspended in 50 ml lysis buffer (50 mM Tris pH 8.0, 5
112
mM EDTA). Cells were sheared with a microfluidizer (3 rounds at 1400 bars, Microfluidics) and
113
crude lysate was clarified by centrifugation (1 h x 50,000 g). Soluble A27 was purified by ion
114
metal affinity chromatography (IMAC) using a HisTrap 5 ml column (GE Healthcare).
115
Supernatant was passed through the HisTrap column and washed with three column volumes (15
116
ml) of wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole). After a final wash at
117
50 mM imidazole, A27 was eluted with the same buffer containing ~ 250 mM imidazole and
118
subsequently dialyzed twice against 10 mM tris pH8.0, 200 mM NaCl prior to size-exclusion
119
chromatography (SEC) for characterization.
120
Protein ELISA. Flat-bottom 96-well microtiter plates were coated with 100 µl of recombinant
121
VACV A27 protein (either C-terminally truncated A27(16-100) or full length A27(16-110) at
122
1 mg/ml, diluted in PBS overnight at 4°C (ThermoScientific Pierce) and washed with washing
123
buffer (PBS, pH 7.2, plus 0.05% Tween 20). Subsequently, plates were blocked with blocking
124
buffer (PBS, pH 7.2, plus 1% BSA plus 0.1% Tween 20) for 2 h at room temperature (RT).
125
Plates were washed and incubated with purified mAb at 10 µg/ml for 90 min at RT. Plates were
126
washed, and the bound mAb was detected by adding a streptavidin-HRP-conjugated secondary
127
antibody to mouse immunoglobulin G (Invitrogen) and incubated for 60 min at RT, followed by
128
OPD substrate (Sigma-Aldrich).
129
Cross-Blocking ELISA. Recombinant A27(16-110) was prepared at 0.5 µg/ml and used to coat
130
Nunc Polysorbent flat-bottom 96-well plates with 100 µl per well. Plates were incubated
131
overnight at 4°C and subsequently washed four times with PBS plus 0.05% Tween 20. 100 µl of
132
blocking buffer (PBS + 10% fetal bovine serum) was added to each well of the plate and the
133
plate was blocked for 90 min at room temperature. Blocking buffer was discarded, and 100 µl of 6
134
purified and unmodified antibodies of interest (at 10 µg/ml) were added to the plate and
135
incubated for 90 min to allow for binding to recombinant A27. Horseradish peroxidase (HRP)-
136
conjugated antibodies of interest (Innova Biosciences Lightning-Link HRP conjugation kit) were
137
prepared at 0.5 µg/ml and added to the plates for 20 min without prior washing of the plate. The
138
ability of the conjugated antibody to bind to A27 in the presence of a pre-bound antibody was
139
assessed using an optical assay. The plates were developed using o-phenylenediamine (OPD),
140
and optical density (OD) at 490 nm was read on a SpectraMax 250 (Molecular Devices).
141
Flow Cytometry-based in vitro neutralization. Vero E6 cells (1x105 cells/well) were seeded in
142
96-well Costar plates (Corning Inc., Corning, NY) and incubated for 5 h to adhere. Subsequently,
143
cells were infected with 12.5 µL purified VACV-GFP at 1x106 PFU/mL (final PFU of 1.25x104)
144
and 12.5 µL mAbs at 80 µg/mL (final concentration of 20 µg/mL) for 12 h at 37°C and 5% CO2
145
in a total volume of 50 µL in the presence (2% final concentration) or absence of sterile baby
146
rabbit complement (CEDARLANE). Samples were prepared in duplicates. Cells were
147
subsequently tested using flow cytometry as described previously (1, 15).
148
Vaccinia intravenous infection protection studies. To infect mice, 1x105 PFU of
149
VACVACAM2000 were injected retro-orbitally. After infecting animals on day 0, weights were
150
taken on day 0 for the initial body weight measurement. Beginning three days post-infection,
151
body weights were taken every other day. Body weights were recorded until the animal reached
152
75% of initial body weight, or if other external health variables became present for which
153
euthanasia was the only humane course of action. Clinical score, a composite score of the pox
154
lesion abundance on the four paws plus the tail, was evaluated as described (22). For A27 mAb
155
protection studies, mice were inoculated i.p. with 100 µg of antibodies 1 day before infection.
156
Control mice received PBS. An additional group received anti-H3 #41 as positive control. 7
157
Animal husbandry and experimental procedures were approved by the Department of Laboratory
158
Animal Care and the Animal Care Committee of the La Jolla Institute
159
Epitope Mapping by Peptide ELISA. Overlapping 20-mer peptides for the A27 antigen were
160
synthesized (AnaSpec) and tested for mAb binding using an ELISA. Flat-bottom 96-well
161
microtiter plates were coated with 100 µl of NeutrAvidin biotin-binding protein (1 mg/ml),
162
diluted in PBS overnight at 4°C (ThermoScientific Pierce) and washed with washing buffer (PBS,
163
pH 7.2, plus 0.05% Tween 20). Subsequently, plates were blocked with blocking buffer (PBS,
164
pH 7.2, plus 1% BSA plus 0.1% Tween 20) for 2 h at room temperature (RT). Plates were
165
incubated with 100 µl of overlapping linear biotinylated peptides (200 ng/ml) in blocking buffer
166
for 90 min at RT. Plates were washed and incubated with purified mAb at 10 µg/ml for 90 min at
167
RT. Plates were washed, and the bound mAb was detected by adding a streptavidin-HRP-
168
conjugated secondary antibody to mouse immunoglobulin G (Invitrogen) and incubated for 60
169
min at RT, followed by OPD substrate (Sigma-Aldrich).
170
Peptide Truncation and Alanine Scan. Variant peptides with N- or C-terminal truncations
171
and/or alanine substitutions were tested for their ability to block binding to the parent 20-mer
172
peptides in ELISA. In case the peptide in question contained an alanine, it was substituted for a
173
serine instead. 96-well plates were coated with 100 µL NeutrAvidin per well at a concentration
174
of 0.5 µg/mL. Plates were incubated overnight at 4°C and washed 4 times with PBS plus 0.05%
175
Tween-20. 100 µL of blocking buffer (PBS + 10% FBS) were added to the plates and incubated
176
for 90 min at 4°C. Subsequently, blocking buffer was discarded, 100 µL of biotinylated 20-mer
177
peptides were added to the plate at 200 ng/mL and incubated for 90 min at 4°C. Simultaneously,
178
selected antibodies were incubated with variant peptides. We used 30 µL/well of mAb at 600
179
ng/mL and incubated it with 30 µL/well of alanine-modified peptides at 100 µg/mL for 90 min at 8
180
4°C. After washing the plates, 50 µL of the antibody/alanine peptide mix was added to plate
181
bound peptides and incubated for 20 min at 4°C. Plates were washed and 100 µL/well of
182
secondary goat anti-mouse anti IgGγ HRP (1:1000 diluted in blocking buffer) were added and
183
incubated for 90 min at 4°C. A final wash step was performed and plates were developed using
184
o-phenylenediamine and OD at 490nm was read on a SpectraMax 250 (Molecular Device).
185
Antibody sequencing. Total RNA from 300 µL hybridoma cells in solution was isolated using
186
the NucleoSpin® RNA II kit according to manufacturer’s instructions (MACHEREY-NAGEL).
187
cDNA was amplified using the OneStep RT-PCR kit (Qiagen). The reverse transcription PCR
188
was performed using primers 5’MsVHE and 3’Cy1 (for isotype IgG1 mAb 6F11), 3’Cy2c outer
189
(for isotype IgG2a mAbs 1G6, 12G2, 12C3 and 4G5) or 3’Cy2b outer (for isotype IgG2b mAb
190
8H10) for the heavy chains, primers 5’mVkappa and 3’mCĸ for the kappa light chains (1G6,
191
12G2, 8H10, 6F11, 12C3 and 4G5) and primers 5’mVλ1/2 and 3’mCλ outer for the lambda light
192
chain (8E3) (29). The cycling profile was slightly modified from manufacturer’s
193
recommendations and set up as follows: 1 cycle of 30 min at 50°C and 15 min at 95°C; 40 cycles
194
of 30 s at 94°C, 45 s at 60°C (for heavy chains) / 58°C (for light chains), and 55 s at 72°C;
195
followed by 1 cycle of 10 min at 72°C and a 12°C cool down. PCR products were verified by gel
196
electrophoresis with a ~500 bp product for heavy chains and ~450 bp product for light chains.
197
Afterwards, PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and
198
then sequenced by Invitrogen (provided with the respective 5’ primer for heavy and light chains).
199
Sequences include V-D-J regions for heavy chains and V-J regions for light chains. Finally,
200
antibody germ lines were determined using IMGT’s V-Quest service (3).
201
Peptide-Fab complex preparation. Purified mAbs 1G6 and 8E3 (1mg/mL in 50 mM NaOAc
202
pH5.5) were incubated with 4% (1G6) and 2% (8E3) (w/w) activated papain (Sigma #P3125) 9
203
and for 4h at 37ºC in 1X digestion buffer. Papain was activated by incubating 20.80 µl papain
204
with 100 µl 10X digestion buffer (1M NaOAc pH 5.5, 12 mM EDTA) and 100µl cysteine (12.2
205
mg/ml) in a total volume of 1ml for 15 min at 37ºC. The papain digestion was stopped by adding
206
20 mM Iodoacetamide (IAA). Digestion mixtures were dialyzed against PBS for subsequent
207
protein A purification to remove undigested IgG and Fc. The protein A flow-through containing
208
Fab’s were concentrated and purified by size exclusion chromatography on a Superdex S200
209
GL10/300 (GE Healthcare), using 50 mM Hepes, pH7.5, 150mM NaCl as running buffer. Fab’s
210
were incubated with 2x molar excess of corresponding A27 peptides for 1 hour at 4˚C and
211
concentrated using 30 kDa centrifugal filtration units to remove unbound peptides.
212
Crystallization and structure determination. Crystals of the 1G6/A2731-40 complex were
213
grown over several days at 22°C by sitting drop vapor diffusion while mixing 0.5 µl protein (5.2
214
mg/ml) with 0.5 µl precipitant (20 % PEG 4000, 200 sodium phosphate dibasic). Crystals were
215
flash-cooled at 100 K in mother liquor containing 20% glycerol. Crystals of the 8E3/A27101-110
216
complex were grown over several days at 4°C by sitting drop vapor diffusion while mixing 0.5
217
µl protein (14 mg/ml) with 0.5 µl precipitant (10 % PEG 3000, 200 mM magnesium chloride,
218
100 mM cacodylate pH 6.5). Crystals were flash-cooled at 100 K in mother liquor containing 20%
219
glycerol. Diffraction data were collected at the Stanford Synchrotron Radiation Laboratory
220
(SSRL) beamline 11-1 (1G6 Fab) and Advanced Light source beamline 5.0.1 (8E3 Fab),
221
processed with iMosflm and SCALA as part of ccp4 (4, 17). Crystal structure of 8E3 was
222
determined by molecular replacement using PHASER (23) and the Fab of a quorum-quenching
223
antibody (PDB code 2NTF), separated in constant and variable domain. The structure of 1G6
224
was obtained similarly by MR, using the PDB coordinated of 8E3 with the CDR loops removed.
225
The model was rebuilt into σA-weighted 2Fo–Fc and Fo–Fc difference electron density maps 10
226
using the program COOT (8). Peptides were manually built in COOT during later stages of
227
refinement. The 1G6/A2731-40 structure was refined to 1.95 Å to an Rcryst and Rfree of 19.8% and
228
23.6%, respectively. The 8E3/A27101-110 structure was refined to 2.27 Å to an Rcryst and Rfree of
229
20.3% and 22.4%, respectively The quality of the models were examined with the program
230
Molprobity (19).
231 232
Results
233
Generation of anti-A27 mAbs
234
Hybridomas were generated from a mouse that had been infected with a sub-lethal dose
235
of VACV. Among them, 12 secreted antibodies reacted with the wild-type VACV but not with
236
an A27-deletion VACV mutant in an immunofluorescence assay of infected HeLa cells. We
237
decided to further characterize seven of those antibodies (1G6 [IgG2a], 12G2 [IgG2a], 8H10
238
[IgG2b], 6F11 [IgG1], 12C3 [IgG2a], 4G5 [IgG2a] and 8E3 [IgG2a]). Out of these, 6 antibodies
239
bound C-terminally truncated A27(16-100) protein in ELISA, whereas the other antibody (8E3)
240
did not bind A27 (Figure 1A). To assess whether 8E3 binds to the C-terminal extremity of A27,
241
we prepared full-length A27(16-110) and repeated the ELISA with four selected mAbs,
242
including 8E3. All tested antibodies were found to bind the new construct, suggesting that 8E3
243
binds to the C-terminal extremity of A27 (Figure 1B).
244
Cross-blocking ELISA was performed, and antibodies 1G6, 12G2 and 8H10 clustered
245
into one distinct cluster (group I). Additionally, 4G5 and 12C3 formed another, distinct cluster
246
(group III). 6F11 and 8E3 could not be cross-blocked by any of the other mAbs and were
247
assigned group II and IV, respectively (Figure 2).
11
248 249
Group I anti-A27 mAbs neutralize MV in a complement-dependent manner
250
We then tested the ability of the anti-A27 mAbs to interfere with VACV MV infection in
251
an in vitro neutralization assay (Figure 3). Vero E6 cells were incubated overnight with purified
252
VACVWR MV expressing a green fluorescent protein (GFP), in the presence or absence of
253
antibody and complement. The samples were evaluated the following day using a flow
254
cytometer. Group I mAbs (1G6, 12G2 and 8H10) neutralized more than 90% of the viruses at 20
255
µg/ml in the presence of complement. In contrast, group II, III and IV mAbs only neutralize the
256
virus by 20% or less in the presence of complement (Figure 3, bottom). Except for mAb 6F11
257
(IgG1), all antibodies were able to bind complement (IgG2a and IgG2b). None of the tested
258
mAbs was able to neutralize in the absence of complement (Figure 3, top). Anti-L1 mAb
259
M12B9 (15), an antibody know to strongly neutralize (>90%) in a complement-independent
260
manner, was used as positive control.
261 262
Group I anti-A27 mAb protects against vaccinia virus infection
263
Next, anti-A27 mAbs were tested in an in vivo vaccinia protection system. SCID mice
264
were infected with 105 PFU ACAM2000 retro-orbitally (Figure 4). In this model, 1G6 (group I),
265
6F11 (group II) and 8E3 (group IV) were tested for their ability to protect against moribundity,
266
weight loss and pox lesions (‘Clinical Score’) (24). Animals were euthanized at 75% initial body
267
weight (end point criteria). Anti-H3 #41 was used as positive control; at day 49 it provided good
268
protection against weight loss (p=0.0024) (Figure 4A) and mortality (p=0.0008) (Figure 4B)
269
compared to control mice receiving no mAb. Anti-H3 #41 also provided robust protection
12
270
against pox lesions (p90%) w/ complement strong (>90%) w/ complement weak (
1
Linear epitopes in A27 are targets of protective antibodies induced
2
by vaccination against smallpox
3 4
Running title: Protective linear epitopes of vaccinia virus A27
5 6
Thomas Kaever1, Michael H. Matho2, Xiangzhi Meng3, Lindsay Crickard1, Andrew Schlossman2,
7
Yan Xiang3, Shane Crotty1,4, Bjoern Peters1, Dirk M. Zajonc2,5^
8 9 10 11 12 13 14 15 16
1
Division of Vaccine Discovery, 2Division of Cell Biology, La Jolla Institute for Allergy and
Immunology (LJI), La Jolla, CA 92037, USA. 3
Department of Microbiology and Immunology, University of Texas Health Science Center at
San Antonio, San Antonio, TX 78229. 4
Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA
92037, USA 5
Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University,
9000 Ghent, Belgium
17 18
^ Corresponding author:
19
Dirk Zajonc
20 21
Abstract: 225 words
22
Full text: 7,496 words
1
23
Abstract
24
Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax
25
smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and
26
binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their
27
protective capacity and their interaction with A27, we generated and subsequently characterized
28
7 murine monoclonal antibodies (mAbs), which fell into 4 distinct epitope groups. Three groups
29
(I, III and IV) bound to linear peptides, while group II only bound to VACV lysate and
30
recombinant A27, suggesting it recognized a conformational and discontinuous epitope. Only
31
group I antibodies neutralized MV in a complement-dependent manner and protected against
32
VACV challenge, while a group II mAb protected partially but did not neutralize. The epitope
33
for group I mAbs was mapped to a region adjacent to the GAG binding site and suggest that
34
group I mAbs could potentially interfere with cellular adhesion of A27. We have further
35
determined the crystal structure of the neutralizing group I mAb 1G6, as well as the non-
36
neutralizing group IV mAb 8E3 bound to the corresponding linear epitope-containing peptides.
37
Both light and heavy chains of the antibodies are important in binding their antigens. For both
38
antibodies the L1 loop seems to dominate the overall polar interactions with the antigen, while
39
for mAb 8E3, the light chain generally appears to make more contacts with the antigen.
40 41
Importance
42
Vaccinia virus is a powerful model to study antibody responses upon vaccination, since
43
its use as the smallpox vaccine led to the eradication of one of the world’s greatest killers. The
44
immunodominant antigens that elicit the protective antibodies are known, yet for many of these 2
45
antigens little information about their precise interaction with antibodies is available. In an
46
attempt to better understand the interplay between the antibodies and their antigens, we have
47
studied a panel of anti A27 antibodies from generation, functional characterization to the
48
interaction with the epitope using X-ray crystallography. We identified one protective antibody
49
that binds adjacent to the heparan sulfate binding site of A27, likely affecting ligand binding.
50
The analysis of antibody-antigen interaction supports a model in which antibodies that can
51
interfere with the functional activity of the antigen are more likely to confer protection than those
52
that bind at the extremities of the antigen.
53 54 55
Introduction Inoculation with vaccinia virus (VACV) elicits neutralizing antibodies against major
56
antigens, including A27, A33, B5, D8, H3, and L1 on both the extracellular enveloped virus (EV)
57
and the intracellular mature virion (MV or IMV), conferring protection against smallpox (2, 6, 7,
58
16, 26). As a result, wide-spread vaccination against smallpox (variola virus, VARV) led to the
59
first eradication of a viral pathogen from nature (11). Among the major immunodominant
60
antigens of the IMV, A27, H3, and D8 are adhesion molecules that bind to the
61
glycosaminoglycans heparan sulfate (A27 and H3) and chondroitin sulfate (D8) (13, 14, 18, 20).
62
We have previously shown that anti-D8 antibodies can prevent binding of D8 to chondroitin
63
sulfate. Besides its binding to heparan sulfate, little is known about the function of H3 (18).
64
However, human antibodies that target H3 in combination with those that target B5 provide
65
significantly better protection than either antibody by itself and are promising for the treatment
66
of smallpox infections in human (22). Since the general human population lacks protection
3
67
against smallpox due to the cessation of smallpox vaccination, protective antibodies can be used
68
to treat infected patients. While neutralizing anti-A27 antibodies protect against infection, they
69
only represent a minor component of the Dryvax vaccine induced immune response (10).
70
A27 is a homo-trimeric extracellular protein that is attached to the viral membrane by
71
binding to the transmembrane protein A17 through its C-terminal leucine zipper domain
72
(residues 80-101). The GAG binding site is located at the N-terminus, downstream of the signal
73
sequence (residues 21-30) (12, 30). The central region of A27 consists of a coiled coil domain
74
(residues 43-84), which is used to interact with the membrane fusion suppressor protein A26
75
through intermolecular disulfide bond formation (Cys71, Cys72). The crystal structure of an N-
76
terminal fragment of A27, containing the heparan sulfate binding site and coiled coil domain
77
(residues 21-84) had recently been determined, however only the central fragment (residues 47-
78
84) is ordered, suggesting flexibility of the N-terminal GAG binding domain (5). The A27
79
structure illustrates the complexity and antiparallel nature of the A27 homo-trimer, yet structural
80
information about the N-terminal and C-terminal extremities are missing.
81
In this study we have produced a panel of anti-A27 antibodies through immunizing mice
82
with VACV. We have identified 4 antibody groups based on cross-blocking experiments and
83
identified the epitope using a peptide/protein ELISA. Group I, II, and IV antibodies recognized
84
both VACV lysate as well as synthetic peptides, suggesting that the epitope of these antibodies
85
can be recapitulated using linear peptides. We have further determined the crystal structure of a
86
protective antibody 1G6 (group I), and a non-protective antibody 8E3 (group IV) in complex
87
with their respective linear peptide epitopes, which are located at the N- and C-terminal
88
extremities of A27, shedding light onto the structural basis of A27 recognition.
4
89
Materials and Methods
90
Viruses and antibodies. VACVWR stocks were grown on HeLa cells in T175 flasks and
91
infecting at a multiplicity of infection of 0.5. Cells were harvested at 60 h and virus was isolated
92
by rapidly freeze-thawing the cell pellet three times in a volume of 2.3 ml RPMI plus 1% fetal
93
calf serum (FCS). Subsequently, cell debris was removed by centrifugation. Clarified supernatant
94
was frozen at -80°C as stock. VACVWR stocks were titered on Vero cells (~2 x 108 PFU/ml).
95
VACVACAM2000 was obtained from the CDC. Monoclonal antibodies used in the study are: anti-
96
H3 #41, anti-D8 Ab12.1, anti-L1 M12B9, anti-A27 1G6, 12G2, 8H10, 6F11, 4G5, 12C3 and 8E3.
97
Hybridoma generation and characterization.
98
The hybridomas were generated as described previously (32). In brief, a six-week old BALB/c
99
mouse was infected intranasally with 5 × 103 plaque-forming-unit (PFU) of VACV WR. Seven
100
weeks after the infection, the mouse was injected intravenously with 7 × 107 PFU of UV-
101
inactivated WR virus. Three days afterwards, the spleen of the mouse was harvested for
102
hybridoma generation. Hybridomas that secret anti-A27 antibodies were identified with
103
immunofluorescence assays of HeLa cells infected with wild-type or A27-deletion VACV strains
104
(31).
105
chromatography and purity was assessed by reducing and non-reducing SDS PAGE. All
106
experiments were performed using purified antibodies.
107
A27 expression and purification.
108
A27L with a C-terminal hexahistidine tag was cloned into pET15b (Invitrogen) and transformed
109
into CodonPlus BL21 cells (Agilent). After culture growth reached an OD600 of 0.6, A27
110
expression was induced by induction with 1mM IPTG at 37 °C for 4 h. Cells from typically 1 L
IgG’s were purified from hybridoma culture media using protein G affinity
5
111
were spun down (10 min x 4000 g) and resuspended in 50 ml lysis buffer (50 mM Tris pH 8.0, 5
112
mM EDTA). Cells were sheared with a microfluidizer (3 rounds at 1400 bars, Microfluidics) and
113
crude lysate was clarified by centrifugation (1 h x 50,000 g). Soluble A27 was purified by ion
114
metal affinity chromatography (IMAC) using a HisTrap 5 ml column (GE Healthcare).
115
Supernatant was passed through the HisTrap column and washed with three column volumes (15
116
ml) of wash buffer (50 mM Tris pH 8.0, 300 mM NaCl, 20 mM imidazole). After a final wash at
117
50 mM imidazole, A27 was eluted with the same buffer containing ~ 250 mM imidazole and
118
subsequently dialyzed twice against 10 mM tris pH8.0, 200 mM NaCl prior to size-exclusion
119
chromatography (SEC) for characterization.
120
Protein ELISA. Flat-bottom 96-well microtiter plates were coated with 100 µl of recombinant
121
VACV A27 protein (either C-terminally truncated A27(16-100) or full length A27(16-110) at
122
1 mg/ml, diluted in PBS overnight at 4°C (ThermoScientific Pierce) and washed with washing
123
buffer (PBS, pH 7.2, plus 0.05% Tween 20). Subsequently, plates were blocked with blocking
124
buffer (PBS, pH 7.2, plus 1% BSA plus 0.1% Tween 20) for 2 h at room temperature (RT).
125
Plates were washed and incubated with purified mAb at 10 µg/ml for 90 min at RT. Plates were
126
washed, and the bound mAb was detected by adding a streptavidin-HRP-conjugated secondary
127
antibody to mouse immunoglobulin G (Invitrogen) and incubated for 60 min at RT, followed by
128
OPD substrate (Sigma-Aldrich).
129
Cross-Blocking ELISA. Recombinant A27(16-110) was prepared at 0.5 µg/ml and used to coat
130
Nunc Polysorbent flat-bottom 96-well plates with 100 µl per well. Plates were incubated
131
overnight at 4°C and subsequently washed four times with PBS plus 0.05% Tween 20. 100 µl of
132
blocking buffer (PBS + 10% fetal bovine serum) was added to each well of the plate and the
133
plate was blocked for 90 min at room temperature. Blocking buffer was discarded, and 100 µl of 6
134
purified and unmodified antibodies of interest (at 10 µg/ml) were added to the plate and
135
incubated for 90 min to allow for binding to recombinant A27. Horseradish peroxidase (HRP)-
136
conjugated antibodies of interest (Innova Biosciences Lightning-Link HRP conjugation kit) were
137
prepared at 0.5 µg/ml and added to the plates for 20 min without prior washing of the plate. The
138
ability of the conjugated antibody to bind to A27 in the presence of a pre-bound antibody was
139
assessed using an optical assay. The plates were developed using o-phenylenediamine (OPD),
140
and optical density (OD) at 490 nm was read on a SpectraMax 250 (Molecular Devices).
141
Flow Cytometry-based in vitro neutralization. Vero E6 cells (1x105 cells/well) were seeded in
142
96-well Costar plates (Corning Inc., Corning, NY) and incubated for 5 h to adhere. Subsequently,
143
cells were infected with 12.5 µL purified VACV-GFP at 1x106 PFU/mL (final PFU of 1.25x104)
144
and 12.5 µL mAbs at 80 µg/mL (final concentration of 20 µg/mL) for 12 h at 37°C and 5% CO2
145
in a total volume of 50 µL in the presence (2% final concentration) or absence of sterile baby
146
rabbit complement (CEDARLANE). Samples were prepared in duplicates. Cells were
147
subsequently tested using flow cytometry as described previously (1, 15).
148
Vaccinia intravenous infection protection studies. To infect mice, 1x105 PFU of
149
VACVACAM2000 were injected retro-orbitally. After infecting animals on day 0, weights were
150
taken on day 0 for the initial body weight measurement. Beginning three days post-infection,
151
body weights were taken every other day. Body weights were recorded until the animal reached
152
75% of initial body weight, or if other external health variables became present for which
153
euthanasia was the only humane course of action. Clinical score, a composite score of the pox
154
lesion abundance on the four paws plus the tail, was evaluated as described (22). For A27 mAb
155
protection studies, mice were inoculated i.p. with 100 µg of antibodies 1 day before infection.
156
Control mice received PBS. An additional group received anti-H3 #41 as positive control. 7
157
Animal husbandry and experimental procedures were approved by the Department of Laboratory
158
Animal Care and the Animal Care Committee of the La Jolla Institute
159
Epitope Mapping by Peptide ELISA. Overlapping 20-mer peptides for the A27 antigen were
160
synthesized (AnaSpec) and tested for mAb binding using an ELISA. Flat-bottom 96-well
161
microtiter plates were coated with 100 µl of NeutrAvidin biotin-binding protein (1 mg/ml),
162
diluted in PBS overnight at 4°C (ThermoScientific Pierce) and washed with washing buffer (PBS,
163
pH 7.2, plus 0.05% Tween 20). Subsequently, plates were blocked with blocking buffer (PBS,
164
pH 7.2, plus 1% BSA plus 0.1% Tween 20) for 2 h at room temperature (RT). Plates were
165
incubated with 100 µl of overlapping linear biotinylated peptides (200 ng/ml) in blocking buffer
166
for 90 min at RT. Plates were washed and incubated with purified mAb at 10 µg/ml for 90 min at
167
RT. Plates were washed, and the bound mAb was detected by adding a streptavidin-HRP-
168
conjugated secondary antibody to mouse immunoglobulin G (Invitrogen) and incubated for 60
169
min at RT, followed by OPD substrate (Sigma-Aldrich).
170
Peptide Truncation and Alanine Scan. Variant peptides with N- or C-terminal truncations
171
and/or alanine substitutions were tested for their ability to block binding to the parent 20-mer
172
peptides in ELISA. In case the peptide in question contained an alanine, it was substituted for a
173
serine instead. 96-well plates were coated with 100 µL NeutrAvidin per well at a concentration
174
of 0.5 µg/mL. Plates were incubated overnight at 4°C and washed 4 times with PBS plus 0.05%
175
Tween-20. 100 µL of blocking buffer (PBS + 10% FBS) were added to the plates and incubated
176
for 90 min at 4°C. Subsequently, blocking buffer was discarded, 100 µL of biotinylated 20-mer
177
peptides were added to the plate at 200 ng/mL and incubated for 90 min at 4°C. Simultaneously,
178
selected antibodies were incubated with variant peptides. We used 30 µL/well of mAb at 600
179
ng/mL and incubated it with 30 µL/well of alanine-modified peptides at 100 µg/mL for 90 min at 8
180
4°C. After washing the plates, 50 µL of the antibody/alanine peptide mix was added to plate
181
bound peptides and incubated for 20 min at 4°C. Plates were washed and 100 µL/well of
182
secondary goat anti-mouse anti IgGγ HRP (1:1000 diluted in blocking buffer) were added and
183
incubated for 90 min at 4°C. A final wash step was performed and plates were developed using
184
o-phenylenediamine and OD at 490nm was read on a SpectraMax 250 (Molecular Device).
185
Antibody sequencing. Total RNA from 300 µL hybridoma cells in solution was isolated using
186
the NucleoSpin® RNA II kit according to manufacturer’s instructions (MACHEREY-NAGEL).
187
cDNA was amplified using the OneStep RT-PCR kit (Qiagen). The reverse transcription PCR
188
was performed using primers 5’MsVHE and 3’Cy1 (for isotype IgG1 mAb 6F11), 3’Cy2c outer
189
(for isotype IgG2a mAbs 1G6, 12G2, 12C3 and 4G5) or 3’Cy2b outer (for isotype IgG2b mAb
190
8H10) for the heavy chains, primers 5’mVkappa and 3’mCĸ for the kappa light chains (1G6,
191
12G2, 8H10, 6F11, 12C3 and 4G5) and primers 5’mVλ1/2 and 3’mCλ outer for the lambda light
192
chain (8E3) (29). The cycling profile was slightly modified from manufacturer’s
193
recommendations and set up as follows: 1 cycle of 30 min at 50°C and 15 min at 95°C; 40 cycles
194
of 30 s at 94°C, 45 s at 60°C (for heavy chains) / 58°C (for light chains), and 55 s at 72°C;
195
followed by 1 cycle of 10 min at 72°C and a 12°C cool down. PCR products were verified by gel
196
electrophoresis with a ~500 bp product for heavy chains and ~450 bp product for light chains.
197
Afterwards, PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and
198
then sequenced by Invitrogen (provided with the respective 5’ primer for heavy and light chains).
199
Sequences include V-D-J regions for heavy chains and V-J regions for light chains. Finally,
200
antibody germ lines were determined using IMGT’s V-Quest service (3).
201
Peptide-Fab complex preparation. Purified mAbs 1G6 and 8E3 (1mg/mL in 50 mM NaOAc
202
pH5.5) were incubated with 4% (1G6) and 2% (8E3) (w/w) activated papain (Sigma #P3125) 9
203
and for 4h at 37ºC in 1X digestion buffer. Papain was activated by incubating 20.80 µl papain
204
with 100 µl 10X digestion buffer (1M NaOAc pH 5.5, 12 mM EDTA) and 100µl cysteine (12.2
205
mg/ml) in a total volume of 1ml for 15 min at 37ºC. The papain digestion was stopped by adding
206
20 mM Iodoacetamide (IAA). Digestion mixtures were dialyzed against PBS for subsequent
207
protein A purification to remove undigested IgG and Fc. The protein A flow-through containing
208
Fab’s were concentrated and purified by size exclusion chromatography on a Superdex S200
209
GL10/300 (GE Healthcare), using 50 mM Hepes, pH7.5, 150mM NaCl as running buffer. Fab’s
210
were incubated with 2x molar excess of corresponding A27 peptides for 1 hour at 4˚C and
211
concentrated using 30 kDa centrifugal filtration units to remove unbound peptides.
212
Crystallization and structure determination. Crystals of the 1G6/A2731-40 complex were
213
grown over several days at 22°C by sitting drop vapor diffusion while mixing 0.5 µl protein (5.2
214
mg/ml) with 0.5 µl precipitant (20 % PEG 4000, 200 sodium phosphate dibasic). Crystals were
215
flash-cooled at 100 K in mother liquor containing 20% glycerol. Crystals of the 8E3/A27101-110
216
complex were grown over several days at 4°C by sitting drop vapor diffusion while mixing 0.5
217
µl protein (14 mg/ml) with 0.5 µl precipitant (10 % PEG 3000, 200 mM magnesium chloride,
218
100 mM cacodylate pH 6.5). Crystals were flash-cooled at 100 K in mother liquor containing 20%
219
glycerol. Diffraction data were collected at the Stanford Synchrotron Radiation Laboratory
220
(SSRL) beamline 11-1 (1G6 Fab) and Advanced Light source beamline 5.0.1 (8E3 Fab),
221
processed with iMosflm and SCALA as part of ccp4 (4, 17). Crystal structure of 8E3 was
222
determined by molecular replacement using PHASER (23) and the Fab of a quorum-quenching
223
antibody (PDB code 2NTF), separated in constant and variable domain. The structure of 1G6
224
was obtained similarly by MR, using the PDB coordinated of 8E3 with the CDR loops removed.
225
The model was rebuilt into σA-weighted 2Fo–Fc and Fo–Fc difference electron density maps 10
226
using the program COOT (8). Peptides were manually built in COOT during later stages of
227
refinement. The 1G6/A2731-40 structure was refined to 1.95 Å to an Rcryst and Rfree of 19.8% and
228
23.6%, respectively. The 8E3/A27101-110 structure was refined to 2.27 Å to an Rcryst and Rfree of
229
20.3% and 22.4%, respectively The quality of the models were examined with the program
230
Molprobity (19).
231 232
Results
233
Generation of anti-A27 mAbs
234
Hybridomas were generated from a mouse that had been infected with a sub-lethal dose
235
of VACV. Among them, 12 secreted antibodies reacted with the wild-type VACV but not with
236
an A27-deletion VACV mutant in an immunofluorescence assay of infected HeLa cells. We
237
decided to further characterize seven of those antibodies (1G6 [IgG2a], 12G2 [IgG2a], 8H10
238
[IgG2b], 6F11 [IgG1], 12C3 [IgG2a], 4G5 [IgG2a] and 8E3 [IgG2a]). Out of these, 6 antibodies
239
bound C-terminally truncated A27(16-100) protein in ELISA, whereas the other antibody (8E3)
240
did not bind A27 (Figure 1A). To assess whether 8E3 binds to the C-terminal extremity of A27,
241
we prepared full-length A27(16-110) and repeated the ELISA with four selected mAbs,
242
including 8E3. All tested antibodies were found to bind the new construct, suggesting that 8E3
243
binds to the C-terminal extremity of A27 (Figure 1B).
244
Cross-blocking ELISA was performed, and antibodies 1G6, 12G2 and 8H10 clustered
245
into one distinct cluster (group I). Additionally, 4G5 and 12C3 formed another, distinct cluster
246
(group III). 6F11 and 8E3 could not be cross-blocked by any of the other mAbs and were
247
assigned group II and IV, respectively (Figure 2).
11
248 249
Group I anti-A27 mAbs neutralize MV in a complement-dependent manner
250
We then tested the ability of the anti-A27 mAbs to interfere with VACV MV infection in
251
an in vitro neutralization assay (Figure 3). Vero E6 cells were incubated overnight with purified
252
VACVWR MV expressing a green fluorescent protein (GFP), in the presence or absence of
253
antibody and complement. The samples were evaluated the following day using a flow
254
cytometer. Group I mAbs (1G6, 12G2 and 8H10) neutralized more than 90% of the viruses at 20
255
µg/ml in the presence of complement. In contrast, group II, III and IV mAbs only neutralize the
256
virus by 20% or less in the presence of complement (Figure 3, bottom). Except for mAb 6F11
257
(IgG1), all antibodies were able to bind complement (IgG2a and IgG2b). None of the tested
258
mAbs was able to neutralize in the absence of complement (Figure 3, top). Anti-L1 mAb
259
M12B9 (15), an antibody know to strongly neutralize (>90%) in a complement-independent
260
manner, was used as positive control.
261 262
Group I anti-A27 mAb protects against vaccinia virus infection
263
Next, anti-A27 mAbs were tested in an in vivo vaccinia protection system. SCID mice
264
were infected with 105 PFU ACAM2000 retro-orbitally (Figure 4). In this model, 1G6 (group I),
265
6F11 (group II) and 8E3 (group IV) were tested for their ability to protect against moribundity,
266
weight loss and pox lesions (‘Clinical Score’) (24). Animals were euthanized at 75% initial body
267
weight (end point criteria). Anti-H3 #41 was used as positive control; at day 49 it provided good
268
protection against weight loss (p=0.0024) (Figure 4A) and mortality (p=0.0008) (Figure 4B)
269
compared to control mice receiving no mAb. Anti-H3 #41 also provided robust protection
12
270
against pox lesions (p90%) w/ complement strong (>90%) w/ complement weak (
