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CORONAVIRUS
Mosaic nanoparticles elicit cross-reactive immune
responses to zoonotic coronaviruses in mice
Alexander A. Cohen
1
, Priyanthi N. P. Gnanapragasam
1
, Yu E. Lee
1
, Pauline R. Hoffman
1
, Susan Ou
1
,
Leesa M. Kakutani
1
, Jennifer R. Keeffe
1
, Hung-Jen Wu
2
, Mark Howarth
2
, Anthony P. West
1
,
Christopher O. Barnes
1
, Michel C. Nussenzweig
3
, Pamela J. Bjorkman
1
*
Protection against severe acute respiratory syndrome c
oronavirus 2 (SARS-CoV-2) a
nd SARS-related emergent
zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding
domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2
RBD along with RBDs from animal betacoronaviruses
that represent threats to humans (mosaic nanoparticl
es with four to eight disti
nct RBDs). Mice immunized
with RBD nanoparticles, but not soluble
antigen, elicited cross-reactive bi
nding and neutrali
zation responses.
Mosaic RBD nanoparticles elicited antibodies with supe
rior cross-reactive recognition of heterologous RBDs
relative to sera from immunizations with homotypic SARS-CoV-2
RBD nanoparticles or COVID-19 convalescent
human plasmas. Moreover, after priming, sera from mosaic RBD
immunized mice neutralized heterologous
pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2
RBD nanoparticle
immunizations, demonstrating no loss of immunogenicity
against particular RBDs resulting from co-display. A
single immunization with mosaic RBD nanoparticles prov
ides a potential strategy to simultaneously protect
against SARS-CoV-2 and emerging zoonotic coronaviruses.
S
evere acute respiratory syndrome coro-
navirus 2 (SARS-CoV-2), a newly emer-
gent betacoronavirus, resulted in a global
pandemic in 2020, infecting millions and
causing the respiratory disease COVID-19
(
1
,
2
). Two other zoonotic betacoronaviruses,
SARS-CoV and Middle East respiratory syn-
drome coronavirus (MERS-CoV), have also re-
sulted in outbreaks within the past 20 years
(
3
). All three viruses presumably originated in
bats (
4
), with SARS-CoV and MERS-CoV adapt-
ing to intermediary animal hosts before jumping
to humans. SARS-like viruses circulate in bats,
and serological surveillance of people living near
caves where bats carry diverse coronaviruses
demonstrates direct transmission of SARS-like
viruses with pandemic potential (
5
). This find-
ing suggests that a pan-coronavirus vaccine is
needed to protect against future outbreaks and
pandemics. In particular, the bat WIV1 and
SHC014 strains are thought to represent an
ongoing threat to humans (
6
,
7
).
Most current SARS-CoV-2 vaccine candi-
dates include the spike trimer (S), the viral
protein that mediates target cell entry after
one or more of its receptor binding domains
(RBDs) adopts an
up
position to bind a host
receptor (Fig. 1A). The RBDs of human corona-
viruses SARS-CoV-2, SARS-CoV, and HCoV-NL63,
as well as those of the related animal corona-
viruses WIV1 and SHC014, use angiotensin-
converting enzyme 2 (ACE2) as their host
receptor (
1
,
8
,
9
), whereas other coronaviruses
use receptors such as dipeptidyl peptidase 4
(
10
)orsialicacids(
11
,
12
). Consistent with its
function in viral entry, S is the primary tar-
get of neutralizing antibodies (
13
22
), with
many targeting the RBD (
14
18
,
21
26
).
Multivalent display of antigen enhances B
cell responses and can p
rovide longer-lasting
immunity than monovalent antigens (
27
,
28
);
thus, protein-based vaccine candidates often
involve a nanoparticle that enables antigen
multimerization. Ma
ny nanoparticles and
coupling strategies have been explored for
vaccine design (
29
), with
plug and display
strategies being especially useful (
30
,
31
). In
one such approach, multiple copies of an en-
gineered protein domain called SpyCatcher
fused to subunits of a virus-like particle form
spontaneous isopeptide bonds to purified
antigens tagged with a 13-residue SpyTag
(
29
32
). The SpyCatcher-SpyTag system was
used to prepare multimerized SARS-CoV-2
RBD or S trimer that elicited high titers of
neutralizing antibodies (
33
,
34
). Although
promising for protection against SARS-CoV-2,
coronavirus reservoirs in bats suggest future
cross-species transmission (
6
,
7
,
35
), necessi-
tating a vaccine that protects against emerging
coronaviruses as well as SARS-CoV-2. Here,
we prepared SpyCatcher003-mi3 nanoparticles
(
31
,
36
) simultaneously displaying SpyTagged
RBDs from human and animal coronaviruses
to evaluate whether mosaic particles can elicit
cross-reactive antibody responses, as previously
demonstrated for influenza head domain
mosaic particles (
37
). We show that mice
immunized with homotypic or mosaic nano-
particles produced broad binding and neu-
tralizing responses, in contrast to plasma
antibodies elicited in humans by SARS-CoV-2
infection. Moreover, relative to homotypic
SARS-CoV-2 nanopartic
les, mosaic nanopar-
ticles showed enhanced heterologous binding
and neutralization properties against hu-
man and bat SARS-like betacoronaviruses
(sarbecoviruses).
We used a study of sarbecovirus RBD re-
ceptor usage and cell tropism (
38
) to guide
our choice of RBDs for co-display on mosaic
particles. From 29 RBDs that were classified
into distinct clades (clades 1, 2, 1/2, and 3)
(
38
), we identified diverse RBDs from SARS-
CoV, WIV1, and SHC014 (clade 1); SARS-CoV-2
(clade 1/2); Rs4081, Yunnan 2011 (Yun11), and
Rf1 (clade 2); and BM-4831 (clade 3). Of these,
SARS-CoV-2 and SARS-CoV are human coro-
naviruses and the rest are bat viruses originat-
ing in China or Bulgaria (BM-4831). We also
included RBDs from the GX pangolin clade 1/2
coronavirus (referred to here as pang17) (
39
);
RaTG13, the bat clade 1/2 virus most closely
related to SARS-CoV-2 (
40
); RmYN02, a clade
2 bat virus from China (
41
); and BtKY72, a
Kenyan bat clade 3 virus (
42
). Mapping of the
sequence conservation
across selected RBDs
showed varying degrees of sequence identity
(68 to 95%), with highest sequence variability
in residues corresponding to the SARS-CoV-2
ACE2 receptor binding motif (Fig. 1, A to D, and
fig. S1). We chose 8 of the 12 RBDs as sources for
three types of mosaic nanoparticles
mosaic-
4a (coupled to SARS-2, RaTG13, SHC014, and
Rs4081 RBDs); mosaic-4b (coupled to pang17,
RmYN02, Rf1, and WIV1 RBDs); and mosaic-
8 (coupled to all eight RBDs)
and compared
them with homotypic mi3 particles constructed
from SARS-CoV-2 RBD alone (homotypic
SARS-2). RBDs from SARS, Yun11, BM-4831,
andBtKY72,whichwerenotcoupledtomosaic
particles, were used to evaluate sera for cross-
reactive responses.
SpyTag003-RBDs were coupled to SpyCatcher003-
mi3 (60 potential conjugation sites) (
36
,
43
)
to make homotypic and mosaic nanoparticles
(Fig. 2A). Particles were purified by size ex-
clusion chromatography (SEC) and analyzed by
SDS
polyacrylamide gel electrophoresis (PAGE),
revealing monodisperse SEC profiles and nearly
100% conjugation (Fig. 2, B and C). Representative
RBDs were conjugated to SpyCatcher003-mi3
with similar or identical efficiencies (fig. S2), which
suggests that mosaic particles contained approxi-
mately equimolar mixtures of different RBDs.
We immunized mice with soluble SARS-
CoV-2 spike trimer (SARS-2 S), nanoparticles
displaying only SARS-2 RBD (homotypic
SARS-2), nanoparticles co-displaying RBDs
(mosaic-4a, mosaic-4b, or mosaic-8), or un-
conjugated nanopart
icles (mi3). Immuno-
globulin G (IgG) responses were evaluated
after prime or boost immunizations (Fig. 3A)
by enzyme-linked immunosorbent assay (ELISA)
against SARS-2 S (Fig. 3B) or a panel of RBDs
(Fig.3,CtoF,andfig.S3).Serafromun-
conjugated nanoparticle-immunized animals
(Fig. 3 and fig. S3, black) showed no responses
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Cohen
et al
.,
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371
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1
Division of Biology and Biological Engineering, California
Institute of Technology, Pasadena, CA 91125, USA.
2
Department of Biochemistry, University of Oxford, Oxford
OX1 3QU, UK.
3
Laboratory of Molecular Immunology, The
Rockefeller University, New York, NY 10065, USA.
*Corresponding author. Email: bjorkman@caltech.edu
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Cohen
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.,
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Fig. 1. Properties of RBDs chosen for this study.
(
A
) Left: Structure of SARS-
CoV-2Strimer(PDB6VXX)withoneRBD(dashedcircle)inan
up
position.
Center and right: Sequence conservatio
nof12RBDscalculatedbytheConSurf
Database (
49
) plotted on a surface representation of the RBD structure (PDB 7BZ5).
Epitopes for representatives from defined classes of RBD-binding antibodies (classes
1to4)(
24
)areindicatedbydashedlines.(
B
) Summary of properties of the viral
strains from which the 12 sarbecovirus RBDs were derived. (
C
) Phylogenetic tree of
human and selected other coronaviruses based on RBD protein sequences. Red
shading indicates strains known to use ACE2 as a receptor. (
D
) Heat map showing
percent amino acid sequence identities among 12 sarbecovirus RBDs.
RESEARCH
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above background. Anti
SARS-2 S trimer and
anti
SARS-2 RBD serum responses were sim-
ilar (Fig. 3, B and C), demonstrating that anti-
bodies elicited against RBDs can access their
epitopes on SARS-2 S trimer. We also con-
ducted in vitro neutralization assays using a
pseudotyped virus assay that quantitatively
correlates with authentic virus neutralization
(
44
)forstrainsknowntoinfect293T
ACE2
target cells (SARS-CoV-2, SARS, WIV1, and
SHC104). Neutralization and ELISA titers were
significantly correlated (fig. S4), which implies
that ELISAs are predictive of neutralization
results when viral entry receptor usage pre-
vents accurate pseudotyped neutralization assays.
Mice immunized with soluble SARS-2 S
trimer showed no binding or neutralization
except for autologous responses against SARS-2
after boosting (Fig. 3, C to F, brown bars). By
contrast, sera from RBD nanoparticle
immunized
animals exhibited binding to all RBDs (Fig. 3,
C to F, red, green, yellow, and blue bars; fig.
S3A) and neutralization against all four strains
after boosting (Fig. 3, C to E), consistent with
increased immunogenicities of multimerized
antigen on nanoparticles versus soluble anti-
gen (
27
,
28
). Homotypic SARS-2 nanoparticles,
but not soluble SARS-2 trimer, induced het-
erologous responses to zoonotic RBDs and
neutralization of heterologous coronaviruses
(Fig. 3, D to F). To address whether co-display
of SARS-2 RBD along with other RBDs on
mosaic-4a and mosaic-8 versus homotypic
display of SARS-2 RBD (homotypic SARS-2)
diminished anti
SARS-2 responses, we com-
pared SARS-2
specific ELISA and neutralization
titers for mosaic versus homotypic immuniza-
tions (Fig. 3C); there wer
e no significant differ-
ences in IgG anti
SARS-2 titers for animals
immunized with homotypic (Fig. 3C, red) versus
mosaic nanoparticles (Fig. 3C, green and blue).
Thus, in terms of the magnitude of immune
response against SARS-2, there was no advan-
tage of immunization with a homotypic RBD
nanoparticle versus a mos
aic nanoparticle that
included SARS-2 RBD.
We next compared serum responses against
matchedRBDs(RBDspresentonaninjected
nanoparticle) versus m
ismatched RBDs (RBDs
not present on an inject
ed nanoparticle) (Fig.
3 and fig. S3, gray and red horizontal shading,
respectively). Although SARS-2 RBD was not
presented on mosaic-4b, antibody titers elicited
by mosaic-4b immunization (yellow) were not
significantly different from titers elicited by
matched nanoparticle immunizations [homo-
typic SARS-2 (red), mosaic-4a (green), and
mosaic-8 (blue)], and sera from boosted mosaic-
4b
immunized mice neutralized SARS-2 pseu-
dovirus (Fig. 3C). In other matched versus
mismatched comparison
s, sera showed bind-
ing and neutralization of SHC014 and WIV1
regardless of whether these RBDs were in-
cluded on the injected nanoparticle (Fig. 3D);
this result implies sharing of common epi-
topes among RBDs (Fig. 1A).
In an experiment that demonstrated the ad-
vantages of mosaic versus homotypic SARS-2
nanoparticles, sera from mosaic-8
immunized
mice bound SHC014 and WIV1 RBDs signifi-
cantly better after priming than sera from
homotypic SARS-2
immunized mice and re-
tained better binding to SHC014 RBD after
boosting (Fig. 3D). Thus, the potential in-
creased avidity of the homotypic SARS-2 nano-
particle displaying only one type of RBD over
the mosaic-8 nanoparticles did not confer in-
creased breadth. Moreover, mosaic-8
immunized
and boosted sera were more potent than sera
from homotypic SARS-2
immunized animals
in neutralizing SHC014 and WIV1 (Fig. 3D).
Neutralization of the SHC014 and WIV1
pseudoviruses by mosaic-8 sera suggests that
combining RBDs on a mosaic nanoparticle does
not diminish the immune response against a
particular RBD, as also suggested by ELISA
binding of sera to Rs4081 and RaTG13 (fig.
S3, A and B).
To further address whether RBD nano-
particles elicited antibodies that recognized
totally mismatched strains and SARS-CoV-2
RBD mutants, we evaluated sera for binding
to SARS, Yun11, BM-4831, and BtKY72 RBDs
Cohen
et al
.,
Science
371
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12 February 2021
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Fig. 2. Construction of RBD
nanoparticles.
(
A
) Left: Spy-
Tagged RBDs were attached to
SpyCatcher003-mi3 to make a
homotypic particle and three
mosaic particles. There are
60 potential coupling sites on
mi3; only 10 are shown for
clarity. (
B
) SEC profile showing
separation of RBD nanopar-
ticles and free RBD proteins.
(
C
) Coomassie-stained
SDS-PAGE of RBD-coupled
nanoparticles, free RBD
proteins, and uncoupled
SpyCatcher003-mi3 particles
(SC3-mi3).
5101520
0
500
1000
1500
2000
homotypic SARS-2
mosaic-4b
mosaic-4a
mosaic-8
SARS-2
SARS-2
SHC014
RaTG13
Rs4081
WIV1
Rf1
RmYN02
pang17
mosaic-8
WIV1
Rf1
RmYN02
pang17
SARS-2
SHC014
RaTG13
Rs4081
Homotypic SARS-2
100
75
50
37
kDa
25
20
SA
RS-2 RBD-mi3
SARS-2 RBD
mosaic-4a-mi3
mosaic-4a RBDs
mosaic-4b-mi3
mosaic-4b RBDs
mosaic-8-mi3
mosaic-8 RBDs
SC3-mi3
RBD-mi3
RBD
A
BC
mosaic-4a
mosaic-4b
RBD-SpyTag003
SpyCatcher003
-mi3
mA
280
mL
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C
E
0.0
0.5
1.0
1.5
2.0
NS
0.0
1.0
2.0
3.0
NS
SARS-2 RBD ELISA
After Prime (Day 14)
After Boost (Day 42)
IgG titers (AUC x10
6
)
After Prime (Day 28)
After Boost (Day 42)
SARS-2 pseudovirus neutralization
Neutralization ID
50
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
NS
NS
D
0.0
0.5
1.0
1.5
2.0
After Prime (Day 14)
****
****
NS
p<0.0001
p<0.0001
IgG titers (AUC x10
6
)
SHC014 RBD ELISA
After Boost (Day 56)
**
p=0.051
p=0.0042
A
Prime + Adjuvant
injection
bleed
spleen harvest
Day
0142842
Boost + Adjuvant
56
SARS-2 S
mosaic-4a
mi3
SARS-2
mosaic-4b
mosaic-8
mismatched
matched
0.0
0.5
1.0
1.5
2.0
After Prime (Day 14)
NS
SARS-2 Spike ELISA
B
After Boost (Day 56)
SHC014 pseudovirus
neutralization
10
2
10
3
10
4
10
5
Neutralization ID
50
IgG titers (AUC x10
6
)
p<0.0001
p=0.030
p<0.0001
*
****
****
*
****
NS
p<0.0001
p=0.020
BM-4831 RBD ELISA
After Prime (Day 14)
After Prime (Day 14)
Yun 11 RBD ELISA
IgG titers (AUC x10
6
)
*
****
NS
p<0.0001
p=0.022
*
*
NS
p=0.038
p=0.021
BtKY72 RBD ELISA
After Prime (Day 14)
After Boost (Day 42)
0.0
0.5
1.0
1.5
2.0
After Boost (Day 42)
p=0.0080
p=0.021
**
*
***
p=0.0006
*
NS
p=0.026
NS
After Boost (Day 42)
F
After Prime (Day 14)
WIV1 RBD ELISA
After Boost (Day 56)
WIV1 pseudovirus
neutralization
After Boost (Day 56)
p=0.028
*
***
NS
p=0.0004
p=0.019
SARS pseudovirus neutralization
After Boost (Day 42)
After Prime (Day 28)
NS
2
3
4
5
10
2
10
3
10
4
10
5
10
10
10
10
Neutralization ID
50
p=0.0002
p=0.0086
0.0
0.5
1.0
1.5
2.0
2.5
p=0.016
*
NS
*
p=0.043
SARS RBD ELISA
After Prime (Day 14)
After Boost (Day 42)
IgG titers (
AUC x10
6
)
soluble, n=5
RBD
nanoparticle
n=10
unconjugated, n=5
0.0
0.5
1.0
1.5
*
NS
****
NS
p<0.0001
NS
NS
****
****
NS
p<0.0001
p<0.0001
*
NS
NS
**
***
NS
Fig. 3. RBD nanoparticles induce cross-reactive IgG responses in immu-
nized mice.
Red and gray rectangles below ELISA and neutralization data
represent mismatched strains (red; RBD from that strain was not present on the
immunized particle) or matched strains (gray; RBD was present on the
immunized particle). (
A
) Left: Immunization schedule; adjuvant is AddaVax
(Invivogen). Right: Key for immunizations; number of mice in each cohort is
indicated. (
B
to
F
) Neutralization and/or binding data for serum IgGs for
recognition of (B) SARS-2 spike trimer, (C) SARS-2 RBD and SARS-2
pseudovirus, (D) SHC014 and WIV1 RBDs and corresponding pseudoviruses,
(E) SARS RBD and SARS pseudovirus, and (F) Yun11, BM-4831, and BtKY72
RBDs. Mice were immunized with soluble SARS-CoV-2 S trimer (SARS-2 S; brown
bars) or the following nanoparticles: homotypic SARS-2 (red), mosaic-4a (green),
mosaic-4b (yellow), mosaic-8 (blue), or unconjugated SpyCatcher003-mi3
(mi3; black). ELISA data from serum IgG responses to SARS-2 spike trimer (B) or
RBDs [(C) to (F)] are shown as area under the curve (AUC). For (C) to (E),
neutralization potencies are presented as half-maximal inhibitory dilutions
(ID
50
values) of sera against the pseudoviruses from the indicated coronavirus
strains. Dashed horizontal lines correspond to the lowest dilution representing the
limit of detection. Each dot represents serum from one animal, with means
and SDs for vaccinated cohorts denoted by rectangles and horizontal lines,
respectively. Significant differences between groups linked by horizontal lines are
indicated by asterisks and
P
values. NS, not significant.
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Cohen
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.,
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12 February 2021
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COV IgG
IOMA
COV IgG
IOMA
COV IgG
IOMA
COV IgG
IOMA
COV IgG
IOMA
COV IgG
IOMA
0
1
10
8
2
10
8
3
10
8
AUC
0.001
0.01
0.1
1
10
100
[IgG]
(
μ
g/mL
)
0.001
0.01
0.1
1
10
100
0
2
10
6
4
10
6
6
10
6
8
10
6
[IgG]
(
μ
g/mL
)
RLU
0.001
0.01
0.1
1
10
100
[IgG]
(
μ
g/mL
)
0.001
0.01
0.1
1
10
100
[IgG]
(
μ
g/mL
)
0.001
0.01
0.1
1
10
100
0
2
10
6
4
10
6
6
10
6
8
10
6
[IgG] (
μ
g/mL)
RLU
COV57 IgG
COV107 IgG
COV72 IgG
COV47 IgG
COV28 IgG
COV67 IgG
COV96 IgG
COV134 IgG
COV98 IgG
COV202 IgG
COV95 IgG
COV41 IgG
COV92 IgG
COV88 IgG
COV7 IgG
COV37 IgG
COV42 IgG
IOMA
COV21 IgG
SARS-2 RBD
RaTG13 RBD
SHC014 RBD
WIV1 RBD
0.001
0.01
0.1
1
10
100
[IgG]
(
μ
g/mL
)
Rs4081 RBD
BM-4831 RBD
AB
C
D
E
F
G
SARS-2 RBD
WIV1 RBD
BM-4831 RBD
SHC014 RBD
RaTG13 RBD
Rs4081 RBD
****
****
****
****
****
p<0.0001
p<0.0001
p<0.0001
p<0.0001
p<0.0001
H
Pseudovirus Neutralization IC
50
(μg/mL)
Fig. 4. IgGs from convalescent COVID-19 plasma show little to no cross-
reactive responses.
(
A
to
F
) Plasma IgG (
18
,
24
) responses were evaluated by
ELISA [data shown as binding curves with plasma names (
18
) listed] against
RBDs from (A) SARS-2, (B) RaTG13, (C) SHC014, (D) WIV1, (E) Rs4081, and (F)
BM-4831. Data points are plotted as means ± SD of duplicate measurements.
IOMA, an anti
HIV-1 IgG (
50
), was used as a control. (
G
) ELISA results from (A)
to (F), presented as AUC; each dot represents one plasma sample, with means ±
SD shown as colored bars. Significant differences between groups linked by
horizontal lines are indicated by asterisks and
P
values. (
H
)IC
50
values for
pseudotyped neutralization assays using IgGs from COV7, COV21, and COV72
plasmas (
18
) (evaluated at top concentrations of 1500
m
g/ml) against the
indicated strains. Mean = arithmetic mean IC
50
.
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(Fig. 3, E and F); SARS-2 RBD mutants (fig.
S3C); and MERS-CoV RBD (fig. S3D), as well
as for neutralization in SARS pseudovirus
assays (Fig. 3E). We found no reductions in
SARS-2 RBD binding as a result of mutations
[Y453F, the
Danish mink variant
(
45
), or a
Q493K/Q498Y/P499T triple mutant (
46
)] (fig.
S3C),nobindingofanyelicitedseratoMERS-
CoV RBD (fig. S3D), and higher and more
cross-reactive antibody responses for mosaic
immunizations than for homotypic SARS-2
immunizations (e.g., mosaic-8
primed and
boosted animals showed significantly higher
titers against SARS RBD than did sera from
homotypic SARS-2
immunized mice) (Fig.
3E). After priming, sera from the homotypic
SARS-2
immunized animals did not neutral-
ize SARS, whereas the mosaic-4b and mosaic-8
sera were neutralizing (Fig. 3E), perhaps be-
cause these nanoparticles included WIV1 RBD,
which is related by 95% amino acid identity to
SARS RBD (Fig. 1D). After boosting, SARS-2
and mosaic-4a sera were also neutralizing, al-
though titers were lower than for mosaic-8
immunized animals by a factor of ~4 (Fig. 3E).
ELISA titers against other mismatched RBDs
(Yun11, BM-4831, and BtKY72) were signifi-
cantly higher for sera collected after mosaic-8
priming than for sera from homotypic SARS-2
priming, and heightened binding was retained
after boosting (Fig. 3F). Thus, relative to homo-
typic SARS-2 nanoparti
cles, mosaic nanopar-
ticles (particularly mosaic-8) induce higher
antibody titers against mismatched RBDs, This
is another finding that favors the co-display
approach for inducing broader anti-coronavirus
responses, especially after a single prime.
Using flow cytometry, we investigated the
potential for cross-reactive recognition
specifically, whether B cell receptors on IgG
+
splenic B cells from RBD nanoparticle
boosted
animals could simultaneously recognize RBDs
from SARS-2 and Rs4081 (related by 70% se-
quence identity) (Fig. 1D and fig. S5). Whereas
control animals were negative, all other groups
showed B cells that recognized SARS-2 and
Rs4081 RBDs simultaneously, suggesting the
existence of antibodies that cross-react with
both RBDs (fig. S5E).
To compare antibodies elicited by RBD
nanoparticle im
munization to antibodies eli-
cited by SARS-CoV-2 infection, we repeated
ELISAs against the RBD panel using IgGs
from COVID-19 plasma donors (
47
) (Fig. 4).
Most of the convalescent plasmas showed
detectable binding to SARS-2 RBD (Fig. 4A).
However, binding to other sarbecovirus RBDs
(RaTG13, SHC014, WIV1, Rs4081, and BM-
4831) was significantly weaker than binding
to SARS 2 RBD, with many human plasma
IgGs showing no binding above background
(Fig. 4, B to G). In addition, although con-
valescent plasma IgGs neutralized SARS-CoV-2
pseudoviruses, they sh
owed weak or no neutral-
ization of SARS, SHC014, or WIV1 pseudoviruses
(Fig. 4H). These results are consistent with little
to no cross-reactive recognition of RBDs from
zoonotic coronavirus strains resulting from
SARS-CoV-2 infection in humans.
Our results confirm that multimerization
of RBDs on nanoparticles enhances immuno-
genicity relative to soluble antigen (
33
,
48
). We
found that homotypic SARS-2 nanoparticle
immunization produces IgG responses that
bind zoonotic RBDs and neutralize heterol-
ogous coronaviruses after boosting. By contrast,
soluble SARS-2 S immunization and natural
infection with SARS-CoV-2 resulted in weak
or no heterologous responses in plasmas. Co-
display of SARS-2 RBD along with diverse
RBDs on mosaic nanoparticles showed no dis-
advantages for eliciting
neutralizing antibodies
against SARS-CoV-2 relative to homotypic
SARS-2 nanoparticles; th
erefore, mosaic nano-
particles may represent a candidate vaccine to
protect against COVID-19. Furthermore, relative
to homotypic SARS-2 RBD particles, the mosaic
co-display strategy demonstrated advantages for
eliciting neutralizing antibodies against zoonotic
sarbecoviruses, thus potentially also providing
protection against emerging coronaviruses with
human spillover potential. Neutralization of
matched and mismatched strains was observed
after mosaic priming; hence, a single injection
of a mosaic RBD nanoparticle might be suffi-
cient in a vaccine. Because COVID-19 convales-
cent plasmas showed little to no recognition of
coronavirus RBDs other than SARS-CoV-2,
COVD-19
induced immunity in humans may
not protect against ano
ther emergent corona-
virus. However, the mosaic nanoparticles de-
scribed here could be used as described or easily
adapted so that they present RBDs from newly
discovered zoonotic coronaviruses.
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ACKNOWLEDGMENTS
We thank K. Brune (Genie Biotech) for advice about mi3
production; J. Bloom (Fred Hutchinson) and P. Bieniasz
(Rockefeller University) for neutralization assay reagents;
J. Vielmetter and Caltech
s Beckman Institute Protein Expression
Center for protein production; A. Flyak for help with flow
cytometry; M. Murphy for figures; COVID-19 plasma donors,
B. Coller, S. Schlesinger, and the Rockefeller University Hospital
Clinical Research Support Office and nursing staff; and A. Flyak
and A. DeLaitsch for critical reading of the manuscript.
Funding:
Supported by NIH grant P01-AI138938-S1 (P.J.B. and M.C.N.), the
Caltech Merkin Institute for Translational Research (P.J.B.), a
George Mason University Fast Grant (P.J.B.), and the Medical
Research Council (MR/P001351/1) (M.H.) (this UK-funded award is
part of the EDCTP2 program supported by the European Union).
M.C.N. is a Howard Hughes Medical Institute Investigator.
Author
contributions:
A.A.C., C.O.B., and P.J.B. conceived and designed
experiments; A.A.C., P.N.P.G., Y.E.L., P.R.H., S.O., and L.M.K.
performed experiments; H.-J.W. generated and validated
SpyCatcher003-mi3; M.H. supervised the generation and validation
of SpyCatcher003-mi3; and A.A.C., J.R.K., A.P.W., C.O.B., M.C.N.,
and P.J.B. analyzed data and wrote the paper with contributions
from other authors.
Competing interests:
M.H. is an inventor on a
patent on SpyTag/SpyCatcher (EP2534484) and a patent
application on SpyTag003:SpyCatcher003 (UK Intellectual
Property Office 1706430.4), as well as a SpyBiotech cofounder,
shareholder, and consultant. P.J.B. and A.A.C. are inventors on a
provisional application from the California Institute of Technology
that covers the mosaic nanoparticles described in this work.
Data and materials availability:
All data are available in the main
text or the supplementary materials. Materials are available upon
request to the corresponding author with a signed material
transfer agreement. This work is licensed under a Creative
Commons Attribution 4.0 International (CC BY 4.0) license, which
permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited. To view
a copy of this license, visit https://creativecommons.org/licenses/
by/4.0/. This license does not apply to figures/photos/artwork or
other content included in the article that is credited to a third party;
obtain authorization from the rights holder before using such material.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/371/6530/735/suppl/DC1
Materials and Methods
Figs. S1 to S5
References (
51
58
)
MDAR Reproducibility Checklist
View/request a protocol for this paper from
Bio-protocol
.
12 November 2020; accepted 7 January 2021
Published online 12 January 2021
10.1126/science.abf6840
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et al
.,
Science
371
, 735
741 (2021)
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Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic
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Alexander A. Cohen, Priyanthi N. P. Gnanapragasam, Yu E. Lee, Pauline R. Hoffman, Susan Ou, Leesa M. Kakutani,
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