of 11
Supplementary Information
Designed mosaic nanoparticles enhance cross-
reactive immune responses in mice
Eric Wang
1,8
, Alexander A. Cohen
2,8
, Luis F. Caldera
2,8
, Jennifer R. Keeffe
2
, Annie V.
Rorick
2
, Yusuf M. Aida
2,6
, Priyanthi N.P. Gnanapragasam
2
, Pamela J. Bjorkman
2,*
, and
Arup K. Chakraborty
1,3-5,7,9,*
1
Institute for Medical Engineering and Science, Mass
achusetts Institute of Technology,
Cambridge, MA 02139.
2
Division of Biology and Biological Engineering
2
, California Institute of Technology,
Pasadena, CA 91125.
3
Department of Chemical Engineering, Massachusetts I
nstitute of Technology,
Cambridge, MA 02139.
4
Department of Physics, Massachusetts Institute of T
echnology, Cambridge, MA 02139.
5
Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, MA
02139.
Present address:
6
School of Clinical Medicine, University of Cambridg
e, Hills Rd,
Cambridge, CB2 0SP, UK
7
Ragon Institute of Massachusetts General Hospital,
Massachusetts Institute of
Technology, and Harvard University, Cambridge, MA 0
2139.
8
These authors contributed equally.
9
Lead contact
*Correspondence:
arupc@mit.edu; bjorkman@caltech.edu
Table S1
. Antibodies in the deep mutational scanning data
1-5
used to calculate escape.
Antibody
RBD epitope
class
6
COV2
-
2196
class 1
S2H14
class 1
COV2
-
2165
class 1
COV2
-
2832
class 1
S2E12
class 1
LY
-
CoV016
class 1
REGN10933
class 1
C105
class 1
S2X58
class 2
S2D106
class 2
S2H13
class 2
S2H58
class 2
S2X16
class 2
LY
-
CoV555
class 2
C121
class 2
C144
class 2
C002
class 2
COV2
-
2096
class 2
COV2
-
2050
class 2
COV2
-
2479
class 2
C135
class 3
S2X227
class 3
COV2
-
2499
class 3
REGN10987
class 3
S309
class 3
COV2
-
2130
class 3
C110
class 3
CR3022
class 4
COV2
-
2677
class 4
S2X35
class 4
COV2
-
2094
class 4
S304
class 4
S2H97
class 4
COV2
-
2082
class 4
S2X259
class 4
Table S2
. The 20 RBD positions with highest escapes from cl
ass 1 and 2 anti-RBD
antibodies
6
based on DMS data
1-5
and their assigned amino acid. An assigned amino
acid was selected as the amino acid with the larges
t mean escape fraction relative to the
WA1 amino acid, which also was not a charged-to-hyd
rophobic mutation.
Antibody RBD-epitope class
RBD position with high escape
Assigned amino acid
1
417
Y
1
420
Q
1
449
D
1
455
R
1
456
G
1
460
L
1
472
P
1
473
F
1
475
N
1
476
S
1
484
K
1
485
S
1
486
A
1
487
K
1
489
L
1
496
V
1
498
D
1
500
Q
1
501
A
1
504
L
2
346
D
2
447
D
2
449
K
2
450
T
2
452
R
2
455
G
2
456
A
2
472
P
2
473
F
2
475
N
2
481
K
2
483
R
2
484
R
2
485
K
2
486
P
2
487
K
2
489
V
2
490
K
2
493
K
2
494
R
Table S3
. Designed RBDs with their specific mutations relat
ive to the SARS-CoV-2 WA1
RBD. 3 mutations are in RBD positions with high esc
ape against class 1 antibodies
6
based on DMS data
1-5
(class 1 escape mutations), and 3 mutations are in
RBD positions
with high escape against class 2 antibodies (class
2 escape mutations). RBDs that pass
experimental validation in terms of expression (Fig
ure 3B) and binding to class 3 and 4
antibodies but not class 1 and 2 antibodies (Figure
3D) are bolded.
RBD Pair RBD ID Class 1 escape mutations Class 2 es
cape mutations
1
RBD1
L455R, F486A, N487K
E484R, Y489V, F490K
RBD2
K417Y, A475N, Q498D
G447D, L452R, Q493K
2
RBD3
L455R, F456G, N487K
E484R, F490K, Q493K
RBD4
A475N, Y489L, N501A
Y449K, L452R, F486P
3
RBD5
K417Y, F486A, Q498D
F456A, E484R, S494R
RBD6
L455R, A475N, N487K
I472P, F490K, Q493K
4
RBD7
A475N, F486A, N487K
L452R, E484R, Y489V
RBD8
L455R, F456G, Y473F
G485K, F490K, Q493K
5
RBD9
F486A, N487K, Y489L
Y449K, L452R, E484R
RBD10
L455R, I472P, A475N
F456A, F490K, S494R
Table S4
. Selected sarbecovirus RBDs along with their GenBa
nk
7
accession numbers,
chosen residue numbers based on alignment with the
SARS-CoV-2 WA1 RBD, and
clade. RBDs that passed experimental validation in
terms of expression (Figure 3B) and
binding to class 3 and 4 antibodies but not class 1
and 2 antibodies (Figure 3D) are
bolded. The clade is defined as described in Starr
et al.
8
Virus
Accession
Residue number
Clade
LYRa3
AHX37569.1
310-527
1a
Khosta-2
QVN46569.1
307-522
3
C028
AAV98001.1
306-523
1a
SHC014
QJE50589.1
307-524
1a
BM48-31
YP 003858584.1
310-524
3
BtKY72
APO40579.1
309-526
3
pang17
QIQ54048.1
317-549
1b
RaTG13
QHR63300.2
319-541
1b
Figure S1.
Illustration of how sequences for class 1 escape m
utations were generated.
From the 20 RBD positions with highest escapes agai
nst class 1 anti-RBD antibodies, we
generated all 38760 possible combinations of 6 posi
tions. For each combination, we
further generated all possible ways to divide the 6
positions into 2 groups of 3, of which
there were 10 possible divisions, resulting in 3876
00 sets of positions. For each set, 1
group of 3 positions was assigned to RBD1 (colored
as light blue or pink), and the other
group of 3 positions was assigned to RBD2 (colored
as dark blue or red). RBD1 and
RBD2 would be mutated in their assigned positions t
o the amino acids listed in Table S2.
Figure S2.
The total escape against class 1 and 2 antibodies
for all ~90,000 RBD pairs
that pass screening. The total escape is obtained f
rom DMS experiments
1-5
and antibody
RBD-epitope classes are defined in Barnes et al.
6
Figure S3.
RBD amino acid sequence identities for computationa
lly designed mosaic
RBD-NPs and mosaic-8b
.
Asterisks indicate strains used for mosaic-RBD NPs.
Figure S4. Mosaic-7
COM
immunization in pre-vaccinated mice elicited super
ior
cross-reactive antibody responses.
The mean of mean titers is compared in panels C
and E by Tukey’s multiple comparison test with the
Geisser-Greenhouse correction
calculated using GraphPad Prism, with pairings by v
iral strain. Significant differences
between immunized groups linked by horizontal lines
are indicated by asterisks: p<0.05
= *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****.
Binding responses at day 0 (before NP or other vacc
ine immunizations) showed
significant differences across cohorts in titers el
icited by the pre-vaccinations.
9
To account
for different mean responses at day 0 between cohor
ts, we applied baseline corrections
in Figure 6 (see Methods)
.
Here, binding data are shown as both baseline corr
ected
(panels B and C) and not baseline corrected (panels
D and E).
(A) Left: Schematic of vaccination regimen. Mice we
re pre-vaccinated with mRNA-LNP
encoding WA1 spike and bivalent WA1/BA.5 prior to p
rime and boost immunizations with
RBD-NPs at day 0 and day 28 or an additional WA1/BA
.5 mRNA-LNP immunization at
day 0. Middle: Colors and symbols (squares) used to
identify immunizations (colors) and
matched (filled in), mismatched (not filled in), or
matched to pre-vaccination (half-filled in)
viral strains (squares). Right: numbers and colors
used for sarbecovirus strains within
clades throughout the figure.
(B) ELISA ED
50
binding titers in serum samples from mice
immunized with the indicated immunogens measured at
day 0, day 28, and day 56 against
spike or RBD proteins from the indicated sarbecovir
us strains (numbers and color coding
as in panel A). (C) Mean ELISA titers for each type
of immunization at the indicated days.
Each circle represents the mean ED
50
titers from mice against a single viral strain of
sera
from mice that were immunized with the specified im
munogen (solid circles=matched;
open circles=mismatched; colors for different strai
ns defined in panel A). (D) Mean fold
change in ELISA ED
50
binding titers from day 0 in serum samples from mic
e immunized
with the indicated immunogens measured at day 0, da
y 28, and day 56 against spike or
RBD proteins from the indicated sarbecovirus strain
s (numbers and color coding as in
panel A). (E) Means of fold changes in ELISA titers
for each type of immunization at the
indicated days. Each circle represents the mean fol
d change in ED
50
titers from mice
against a single viral strain of sera from mice tha
t were immunized with the specified
immunogen (solid circles=matched; open circles=mism
atched; colors for different strains
defined in panel A).
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