of 5
Cell Reports, Volume
43
Supplemental information
Human antibodies in Mexico and Brazil
neutralizing tick-borne
fl
aviviruses
Tomás Cervantes Rincón, Tania Kapoor, Jennifer R. Keeffe, Luca Simonelli, Hans-Heinrich
Hoffmann, Marianna Agudelo, Andrea Jurado, Avery Peace, Yu E. Lee, Anna
Gazumyan, Francesca Guidetti, Jasmine Cantergiani, Benedetta Cena, Filippo
Bianchini, Elia Tamagnini, Simone G. Moro, Pavel Svoboda, Federico Costa, Mitermayer
G. Reis, Albert I. Ko, Brian A. Fallon, Santiago Avila-Rios, Gustavo Reyes-
Téran, Charles M. Rice, Michel C. Nussenzweig, Pamela J. Bjorkman, Daniel
Ruzek, Luca Varani, Margaret R. MacDonald, and Davide F. Robbiani
Fig
ure
S
1
.
Gating strategy for sorting
POWV
-
EDIII
-
specific B cells by flow cytometry (related to Fig
ure
2
)
Fig
ure
S
2
.
Lack of TBEV neutralization and
effects of 40ºC incubation
(related to Figure 3)
(
A
)
Lack of TBEV neutralization by P002 and P003, as determined by PRNT.
T
wo independent experiments
(m
ean ±
SD
).
(
B
-
D
)
Incubation at 40
ºC to favor virus breathing does not alter the ability
of P002 and P003 to
neutralize the indicated
flav
iviruses
, as determined by
a
microscopy
-
based neutralization assay.
One experiment, m
ean ± SD of triplicates.
Fig
ure
S
3
.
NMR informs
on the
P003
epitope on
EDIII
(related to Figure 4)
(
A
)
Comparison of the NMR signal of EDIII (grey) with its complex with P003 Fab (green) identifi
es
residues affected
by antibody binding.
(
B
) Residues affected by antibody binding in NMR epitope mapping are shown in green on the surface of ZIKV EDIII
(top, two opposite faces are shown; PDB ID:
5OMZ
) and on the EDIII sequence (bottom). Residues in grey have no
NMR information.
(
C and E
) Binding of P003 to ZIKV
and POWV (lineage I)
EDIII mutated at epitope residues, assessed by ELISA.
Related to Fig.
4
D
and
4G
. Average of two independent experiments.
(
D
) Bi
o
layer interferometry confirms binding of P003 to an
EDIII epitope that is different from those of antibodies Z015
and Z021. Z021 and Z015
(
but not P003 used as negative control
)
, bind to ZIKV EDIII immobilized by P003.
Fig
ure
S
4
.
The P003 epitope on the EDIII is highly conserved across flaviviruses
(related to Figure 5)
(
A
) Alignment of the EDIII sequences of flaviviruses known to cause human disease. Dots indicate P003
contact
residues, and arrow points to
P003 epitope at position 329 (
ZIKV
numbering).
Related to Fig
ure
5
A
.
(
B
)
Alignment of the P003 contact residues with the EDIII of other human
-
infecting flaviviruses
. Similar to Fig
ure
5
A
,
but based on the consensus sequence of each
indicated
flavivirus (see
STAR
Methods).
The residue numbering is
based on ZIKV EDIII.
(
C
) Coldmaps representing on the POWV EDIII the frequency of identical residues at the P003 epitope across the
flaviviruses recognized by P003
.
Similar
to Fig
ure
5
B
, but based on the consensus sequence of each indicated
flavivirus; r
elated to
panel
B
.