Immunity, Volume
50
Supplemental Information
Broad and Potent Neutralizing Antibodies
Recognize the Silent Face of the HIV Envelope
Till Schoofs, Christopher O. Barnes, Nina Suh-Toma, Jovana Golijanin, Philipp
Schommers, Henning Gruell, Anthony P. West Jr., Franziska Bach, Yu Erica Lee, Lilian
Nogueira, Ivelin S. Georgiev, Robert T. Bailer, Julie Czartoski, John R. Mascola, Michael S.
Seaman, M. Juliana McElrath, Nicole A. Doria-Rose, Florian Klein, Michel C.
Nussenzweig, and Pamela J. Bjorkman
Supplementary Figures
Figure S1. Polyreactivity and
pharmacokinetics
assays of SF12 and SF5 antibodies.
Related to Figure 1.
(A) H
E
p
-
2 assay for potential autoreactivity of indicated bNAbs.
Data representative of 2 repeat assays.
(B) Polyreactivity
ELISA
-
based assay detecting non
-
specific binding of a panel of bNAbs and a control protein (BSA) to a baculovirus
extract. (C)
In vivo
pharmacokine
t
ics
of SF12 IgG compared with 3BNC117 IgG measured in
6
-
week old
non
-
reconstituted NRG
mice
(
3 mice
each
antibody
)
.
One independent experiment. Shown is
Mean
of all 3 mice
± SD.
4
E
1
0
n
e
g
.
c
t
r
l
.
2
F
5
S
F
5
S
F
1
2
0
1
3
6
1
0
1
4
0
.
1
1
1
0
S
F
1
2
3
B
N
C
1
1
7
N
6
N
I
H
4
5
-
4
6
4
5
-
4
6
m
2
P
G
T
1
2
8
4
E
1
0
P
G
1
6
P
G
T
1
2
1
1
0
-
1
0
7
4
2
F
5
B
S
A
S
F
5
2
0
3
0
4
0
5
0
6
0
1
0
1
0
0
5
0
0
D
a
y
s
a
f
t
e
r
i
n
j
e
c
t
i
o
n
P
r
o
t
e
i
n
s
S
e
r
u
m
l
e
v
e
l
(
R
L
U
(
i
n
t
h
o
u
s
a
n
d
s
)
μ
g
/
m
L
)
3
B
N
C
1
1
7
S
F
1
2
A
B
C
5
0
μ
m
5
0
μ
m
5
0
μ
m
5
0
μ
m
5
0
μ
m
Figure S2. Data collection and processing for the SF12
–
B41
–
10
-
1074 complex.
Related to Figure 3. (
A
)
Representative micrograph of SF12
-
B41
-
10
-
1074 complex in vitreous ice with in
dividual particles boxed (orange).
Inset: power spectrum of micrograph determined during CTF estimation showing Thon rings to 3Å. (
B
) Reference
-
free 2D classification of 4x4 binned extracted particles. 2D class averages showing secondary structure were sel
ected
for further processing. (
C
) After 3D auto
-
refinement of ab initio model, good particles were subjected to rounds of
3D classification with C1 symmetry applied. 3D classes that showed similar features were pooled and 3D auto
-
refined
to generate final
reconstructions at 3.28Å and 4.36Å for class 1 and class 2, respectively.
A
C
B
Autopicking
2D Classification
~370k Particles
SF12 Fab
10-1074 Fab
Ab initio
3D classification
k=8
~305k Particles
3D Refine
3D classification
Mask Fab C
H
C
L
k=6
~240k Particles
Mask Fab C
H
C
L
CTF Refinement
Movie Refinement
FSC
0.143
=3.28 Å
~55k Particles
Mask Fab C
H
C
L
CTF Refinement
Movie Refinement
FSC
0.143
=4.43 Å
SF12 Fab
SF12 Fab
10-1074 Fab
10-1074 Fab
SF12 Fab
SF12 Fab
Figure S3. Validation and cryo
-
EM map quality.
Related to Figure 3. (
A
) Fourier shell correlation (FSC) plots
calculated from half
-
maps of masked (red), unmasked (blue), and correc
ted (black) data for Class 1. Dotted lines for
FSC values of 0.5 and 0.143 are shown. (
B
) Angular distribution 3D histogram, (
C
) local resolution estimation
(Resmap), and (
D
) representative density from Fab and Env regions of the Class 1 map.
20
10
6.7
5.0
4.0
3.3
2.9
Resolution (Å)
Masked
Corrected
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
G
o
l
d
S
t
a
n
d
a
r
d
F
S
C
FSC Plots for SF12–B41–10-1074
Unmasked
g
p
4
1
r
e
s
i
d
u
e
s
5
7
3
-
5
9
2
N
3
6
2
g
p
1
2
0
g
l
y
c
a
n
g
p
1
2
0
V
4
l
o
o
p
r
e
s
i
d
u
e
s
3
9
5
-
4
1
0
A
C
D
B
3
.
0
Å
3
.
5
Å
4
.
0
Å
4
.
5
Å
5
.
0
Å
5
.
3
Å
Figure S4. Comparison of SF12 and VRC
-
PG05 footprints on gp120.
Related to Figure 3 and Figure 4. Differences
in CDR loop orientations (ribbon) on gp120 (gray surface) by (
A
) SF12 (magenta, CDRH1
-
3; light pink, CDRL1
-
3)
and (
B
) VRC
-
PG05 (forest
green
, CDRH
1
-
3; light green, CDRL1
-
3). The protein epitope on
the gp120
surface is
highlighted
in orange (SF12) or yellow (VRC
-
PG05)
. The glycan portion of the epitope
was
removed for clarity.
C
D
R
H
1
C
D
R
H
1
C
D
R
L
1
C
D
R
L
1
g
p
1
2
0
g
p
1
2
0
C
D
R
H
2
C
D
R
H
2
C
D
R
L
2
C
D
R
L
2
C
D
R
H
3
C
D
R
H
3
C
D
R
L
3
C
D
R
L
3
A
S
F
1
2
-
g
p
1
2
0
p
r
o
t
e
i
n
c
o
n
t
a
c
t
s
B
V
R
C
-
P
G
0
5
-
g
p
1
2
0
p
r
o
t
e
i
n
c
o
n
t
a
c
t
s
Figure S5. Comparison of SF12 and SF5 clonal variants.
Related to
Figure 2, Figure 5, and Figure 6. (
A
) Sequence
alignment of SF12 and SF5 with deduced germline sequences. SHMs are shown for each antibody with paratope
residues denoted. (
B, C
) Modeling of SF5 residues (olive) onto the SF12
-
Env structure. In each case, S
HMs in SF5
potentially enhance recognition of the Env epitope through hydrogen bonding. (
D
) Modeling of SF5 Y77
HC
onto the
10
-
1074 bound protomer shows clashes between this residue and the slightly shifted N295
gp120
glycan (pale green).
Supplementary
Tables
Table S1. Sequence analysis of silent face antibodies from donor 27845.
Related to Figure 1.
M
C
W
e
l
l
V
-
g
e
n
e
%
n
t
m
u
t
a
t
i
o
n
%
A
A
m
u
t
a
t
i
o
n
C
D
R
H
3
(
A
A
)
C
D
R
H
3
(
s
e
q
)
V
-
g
e
n
e
%
n
t
m
u
t
a
t
i
o
n
%
A
A
m
u
t
a
t
i
o
n
C
D
R
3
(
A
A
)
C
D
R
L
3
(
s
e
q
)
S
F
3
I
G
V
H
4
-
5
9
*
0
1
2
5
.
3
%
3
9
.
2
%
2
3
G
R
V
G
P
G
G
L
F
D
R
W
R
G
Y
H
G
H
K
W
V
D
A
I
G
K
V
3
-
2
0
*
0
1
2
1
.
4
%
2
9
.
0
%
6
Q
Q
Y
G
R
T
S
F
5
I
G
V
H
4
-
5
9
*
0
1
1
9
.
0
%
2
5
.
5
%
2
3
A
R
L
G
P
G
G
I
F
D
R
W
T
G
H
Y
G
D
K
W
L
D
P
I
G
K
V
3
-
2
0
*
0
1
1
6
.
0
%
2
1
.
5
%
6
Q
L
S
G
R
R
S
F
7
I
G
V
H
4
-
5
9
*
0
1
2
5
.
3
%
3
9
.
2
%
2
3
G
R
V
G
P
G
G
L
F
D
R
W
R
G
Y
H
G
H
K
W
V
D
A
I
G
K
V
3
-
2
0
*
0
1
2
1
.
4
%
2
9
.
0
%
6
Q
Q
Y
G
R
T
S
F
2
I
G
V
H
4
-
5
9
*
0
1
2
4
.
4
%
3
2
.
0
%
2
3
A
R
L
G
P
G
G
L
F
D
R
Y
T
G
Y
H
G
R
K
W
L
D
A
I
G
K
V
3
-
2
0
*
0
1
1
8
.
0
%
2
3
.
9
%
6
Q
Q
Y
G
R
T
S
F
8
I
G
V
H
4
-
5
9
*
0
1
2
4
.
4
%
3
2
.
0
%
2
3
A
R
L
G
P
G
G
L
F
D
R
Y
T
G
Y
H
G
R
K
W
L
D
A
I
G
K
V
3
-
2
0
*
0
1
1
6
.
5
%
2
1
.
7
%
6
Q
Q
Y
G
R
T
S
F
1
0
I
G
V
H
4
-
5
9
*
0
1
2
5
.
3
%
3
7
.
1
%
2
3
G
R
V
G
P
G
G
L
F
D
R
W
T
G
Y
H
G
H
K
W
V
D
A
I
G
K
V
3
-
2
0
*
0
1
1
9
.
9
%
2
9
.
0
%
6
Q
Q
Y
G
R
T
S
o
r
t
W
e
l
l
V
-
g
e
n
e
%
n
t
m
u
t
a
t
i
o
n
%
A
A
m
u
t
a
t
i
o
n
C
D
R
H
3
(
A
A
)
C
D
R
H
3
(
s
e
q
)
V
-
g
e
n
e
%
n
t
m
u
t
a
t
i
o
n
%
A
A
m
u
t
a
t
i
o
n
C
D
R
3
(
A
A
)
C
D
R
L
3
(
s
e
q
)
S
F
1
2
I
G
V
H
4
-
5
9
*
0
1
1
7
.
0
%
2
1
.
4
%
2
3
A
R
L
G
P
G
G
I
F
D
R
W
T
G
H
Y
G
D
K
W
L
D
P
I
G
K
V
3
-
2
0
*
0
1
1
4
.
6
%
2
0
.
4
%
6
Q
L
S
G
R
R
S
F
1
0
I
G
V
H
4
-
5
9
*
0
1
2
5
.
3
%
3
7
.
1
%
2
3
G
R
V
G
P
G
G
L
F
D
R
W
T
G
Y
H
G
H
K
W
V
D
A
I
G
K
V
3
-
2
0
*
0
1
1
9
.
9
%
2
9
.
0
%
6
Q
Q
Y
G
R
T
A
n
t
i
b
o
d
i
e
s
c
l
o
n
e
d
f
r
o
m
m
i
c
r
o
c
u
l
t
u
r
e
d
o
u
b
l
e
h
i
t
s
L
i
g
h
t
c
h
a
i
n
H
e
a
v
y
c
h
a
i
n
C
l
o
n
e
m
e
m
b
e
r
s
i
s
o
l
a
t
e
d
b
y
B
G
5
0
5
-
s
o
r
t
i
n
g
u
p
o
n
m
i
c
r
o
c
u
l
t
u
r
e
i
d
e
n
t
i
f
i
c
a
t
i
o
n
o
f
c
l
o
n
e
H
e
a
v
y
c
h
a
i
n
L
i
g
h
t
c
h
a
i
n
Table S2. 119
virus cross
-
clade panel neutralization data
. Related to Figure 1.